The present invention relates to a method of effectively preventing glycation-induced and other damage to proteins, lipids and DNA by scavenging dicarbonyl intermediates with penicillamine, penicillamine derivatives and other xcex1-amino-xcex2,xcex2-mercapto-xcex2,xcex2-dimethyl-ethane derivatives as dicarbonyl trapping agents. The dicarbonyl scavenging activity of this class of compounds renders them useful as therapeutic agents for the prevention of and treatment of conditions associated with reactive carbonyl compounds and photodamage.
Tissue deterioration and aging have long been associated with accumulation of chemical inducted protein and DNA damage. Reactive oxygen species (ROS) and reactive carbonyl species (RCS), especially xcex1-dicarbonyl compounds, are key mediators of damage caused by oxidative stress, glycation, and UV-irradiation. Carbonyl stress additionally originates from the metabolic generation of methylglyoxal. The toxic effects of various mono- (e.g. 4-hydroynonenal) and xcex1-dicarbonyls (e.g. glyoxal, methylglyoxal, deoxyosones) cannot be directly antagonized by antioxidants and only a small number of biological carbonyl scavengers like glutathione (GSH) have been identified.
The nonenzymatic reactivity of biomolecules is generally regarded as a major endogeneous source of damage to cells. Glycation is a nonenzymatic posttranslational modification of proteins by reducing sugars, which adversely affects protein function. These are subsequently converted to advanced glycosylation end products (AGEs) which represent a heterogenous class of reactive products which form spontaneously in vivo due to the reaction of glucose and other reducing sugars with amino groups of proteins in a concentration dependent manner. These undergo further rearrangements, dehydrations and cross-linking with other proteins to form the AGEs which play a role in long term complications of aging and diabetes.
Lipid peroxidation is another deleterious reaction that targets membrane associated lipids by oxidative mechanisms. Damage to proteins, lipids and nucleic acids by the formation and cellular accumulation of AGEs and peroxidation products has been implicated in a number of age-related diseases including long term diabetic complications (see Thorpe, S: R., and J. W. Baynes. 1996. Role of the Maillard reaction in diabetes mellitus and diseases of aging. Drugs Aging. 9:69-77), atherosclerosis (see Ruderman,N. B., J R. Williamson, and M. Brownlee. 1992. Glucose and diabetic vascular disease [published erratum appears in FASEB J 1993 Jan;7(1):237]. FASEB J. 6:2905-2914), Alzheimer""s disease (see Vitek, M. P., K. Bhattacharya, J. M. Glendening, E. Stopa, H. Vlassara, R. Bucala, K. Manogue, and A. Cerami. 1994. Advanced glycation end products contribute to amyloidosis in Alzheimer disease. Proc Natl Acad Sci U SA. 91:4766-4770) chronic inflammation and the general pathology of the aging process (see Frye, E. B., T. P. Degenhardt, S. R. Thorpe, and J. W. Baynes. 1998. Role of the Maillard reaction in aging of tissue proteins. Advanced glycation end product-dependent increase in imidazolium cross-links in human lens proteins. J. Biol Chem. 273:18714-18719). Glycation and lipid peroxidation are characterized by the formation of very reactive, toxic dicarbonyl derivatives such as glyoxal, methylglyoxal, malondialdehyde, and 3-desoxyosones (Thomalley, P. J., Langborg, A., and Minhas, H. S. 1999. Formation of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose. Biochem. J. 344, 109-116). The crucial role of the generation of reactive carbonyl intermediates, especially dicarbonyl compounds, for the above-mentioned pathologies is well established and has led to the elaboration of the carbonyl hypothesis of aging (Yin, D. 1995. Studies on AGE pigments evolving into a new theory of aging. Gerontology 41, 159-172).
The arginine-derived imidazolium AGE-products (Lander, H M et al. Activation of the receptor for advanced glycation end products triggers a p21 (ras)-dependant mitogen-activated protein kinase pathway regulated by oxidant stress. J. Biol. Chem 272:17810-4, 1997), the glyoxal-lysine dimer (GOLD) and the methylglyoxal-lysine dimer (MOLD) (Brinkmann, Frye E et al., Role of Malliard reaction in aging tissue proteins, Advanced glycation end product-dependant increase in imidazolium cross-links in human lens proteins. J. Biol. Chem. 273:18714-18719, 1998) have been identified imaged human lens crystallin and skin collagen implicating alpha-dicarbonyl stress in tissue aging. Additionally, RCS like glyoxal, the direct precursor of the AGE Nxcex5-carboxymethyl-L-lysine (CML), are generated by free radical damage to polyunsaturated fatty acids in cellular membranes (Fu, M. X. et al., The advanced glycation end product, Nepsilon-(carboxymethyl)lysine, is a product of both lipid peroxidation and glycoxidation reactions. J. Biol. Chem.271:9982-6,1996). UV-irradiation is another source of tissue carbonyl stress, as evidenced by the accumulation of CML in sun exposed lesions of actinic elastosis (Mizutari, K. et al., Photo- enhanced modification of human skin elastin in actinic elastosis by N(epsilon)-(carboxymethyl)lysine, one of the glycoxidation products of the Malliard reaction J. Invest. Dermatol. 108:797-802, 1997. Therefore, AGE-products like CML and GOLD may be regarded as biomarkers of tissue carbonyl stress.
Methylglyoxal is an important glycation intermediate (Thornally et al. Biochem J. 344:109-116, 1999), that is also generated as a biological metabolite by nonenzymatic and enzymatic degradation of glycolic triose phosphate intermediates and from threonine catabolism (Thornally, Pharmacology of Methylglyoxal: Formation, Modification of Proteins and Nucleic Acids and Enzymatic Detoxification-A role in Pathogenesis and Antiproliferative Chemotherapy, Gen. Pharmac. 27: 565-573, 1996). Increased levels of methylglyoxal are found in blood from diabetic patients Beisswenger et al. Metformin reduces systemic methylglyoxal levels in type 2 diabetes, Diabetes 48:198-202, 1999. and in the lens of streptozotocin-induced diabetic rats. A recent study on the formation of AGEs in endothelial cells cultured under hyperglycemic conditions indicated that methylglyoxal was the major precursor of AGEs (Shinohara, M. et al., Overexpression of glyoxalase I in bovine endothelial cells inhibits intracellular advanced glycation endproduct formation and prevents hyperglycemia-induced increases in macromolecular endocytosis. J. Clin. Invest. 101:1142-7, 1998). Various methylglyoxal-derived AGEs have been identified in human tissues, such as fluorescent 5-methylimidazolone-derivatives in atherosclerotic lesions of the aorta (Uchida, K. et al. Protein modification by a Malliard reaction intermediate methylglyoxal. Immunochemical detection of fluorescent 5-methylimidazolone derivatives in vivo. FEBS Lett. 410:313-318,1997. or MOLD and Nxcex5-carboxymethyl-L-lysine in aged skin collagen (Brinkmann supra) Recently, the cytotoxic effects of the glycation intermediates methylglyoxal and 3-deoxyglucosone on neuronal cells such as PC12 cells (Suzuki, K et al. Overexpression of aldehyde reductase protects PC12 cells from the cytotoxicity of methylglyoxal or 3-deoxyglucosone, J. Biochem (Tokyo) 123:353-7, 1998) and cultured cortical neurons Kikuchi, S. et al. Neurotoxicity of methylglyoxal and 3-deoxyglucosone on cultured cortical neurons: synergism between glycation and oxidative stress, possibly involved in neurodegenerative diseases. J. Neurosci. Res. 57:280-289, 1999. have attracted considerable attention because of their suspected participation in the pathogenesis of neurodegenerative diseases such as Alzheimer""s disease (Vitek, M. P. et al., Advanced glycation end products contribute to amyloidosis in Alzheimer disease Proc. Natl. Acad. Sci. USA, 91:4766-70,1994) and amyotrophic lateral sclerosis Shinpo, K. et al. Selective vulnerability of spinal motor neurons to reactive dicarbonyl compounds, intermediate products of glycation, in vitro, implication of inefficient glutathione system in spinal motor neurons. Brain Res. 861:151-159, 2000.
As another result of oxidative and carbonyl stress, protein damage by carbonylation has been associated with aging and a number of diseases, such as the premature aging diseases, Progeria and Werner""s syndrome (Berlett, B. S. et al., Protein oxidation in aging, disease and oxidative stress. J. Biol. Chem. 272:20313-20316, 1997). The amount of carbonyl groups in human skin fibroblast proteins strongly correlates with the age of the donor (Oliver, C, N, et al. Age-related changes in oxidized proteins. J. Biol. Chem. 262:5488-5491, 1987). Elevated levels of histone H1 carbonylation in vivo as an indicator of nuclear oxidative and glycoxidative stress have been reported Wondrak, G.T. et al. Histone carbonylation in vivo and in vitro, Biochem J. 351:769-777, 2000.
In contrast with their therapeutic potential, only a very limited number of biological inhibitors of cellular carbonyl stress like the nucleophilic carbonyl scavenger glutathione have been identified. However, some inhibitors of glycation interfere with the reaction by trapping intermediate alpha-carbonyls, whereas other inhibitory substances act merely as antioxidants and transition metal chelators, thereby inhibiting advanced glycoxidation, but not glycation (Elgawish, A et al. Involvement of hydrogen peroxide in collagen cross-linking by high glucose in vitro and in vivo. J. Biol. Chem. 271:12964-71, 1996). Systemic administration of the hydrazine derivative and carbonyl reagent aminoguanidine, a member of the first class of glycation inhibitors, effectively suppresses secondary complications in diabetic rodents with experimental diabetes and inhibits skin collagen crosslinking (Edelstein, D. et al., Mechanistic studies of advanced glycosylation end product by aminoguanidine. Diabetes 41:26-9, 1992; Fu, M. X. et al., Glycation, glycoxidation, and cross-linking of collagen by glucose, Kinetics, mechanisms and inhibition of late stages of the Malliard reaction, Diabetes 43: 676-83, 1994). A nucleophilic bidentate, phenylacylthiazolium bromide, has been shown to protect E. coli against methylglyoxal toxicity Ferguson et al. Detoxification of methylglyoxal by the nucleophilic bidentate, phenylacylthiazolium bromide, Chem. Res. Tox. 12:617-622, 1999). Other nucleophilic compounds acting as carbonyl traps like tenilsetam (Shoda, H et al., Inhibitory effects of tenilsetam on the Malliard reaction. Endocrinology 138:1886-92,1997), pyridoxamine (Onorato, J. M. et al. J. Biol. Chem. 275:21177-21184, 2000) and metformin Ruggerio-Lopez et al. Reaction of metformin with dicarbonyl compounds. Possible implications in the inhibition of advanced glycation end product formation, Biochem. Pharm. 58:1765-1773, 1999) are being evaluated for prevention of secondary diabetic complications.
In vitro-screening for potential alpha-dicarbonyl scavengers is complicated by the nature of most of the currently employed glycoxidative reaction systems, which measure the suppression of oxygen dependent AGE-formation assessed by AGE fluorescence or immunological quantification of specific AGEs like CML. Consequently, in these glycoxidation systems AGE formation is effectively inhibited by compounds with antioxidant and metal chelating activity. Oxygen-independent advanced glycation by pentoses with formation of AGE fluorescence and protein crosslinking has been demonstrated and mechanistically linked to nonoxidative formation of deoxypentoses as reactive alpha-dicarbonyl intermediates Litchfield, J. E. et al. Oxygen is not required for the browning and crosslinking of protein by pentoses: relevance to Malliard reactions in vivo. Int. J. of Biochem. Cell Biol 31:1297-1305, 1999. Based on the identification of an accelerated glycation reaction between the phosphate-substituted pentose ADP-ribose and histone H1, which produces AGEs without involvement of oxygen Wondrak supra, the assay described herein was developed to screen glycation inhibitors acting as carbonyl scavengers.
It is an object of the present invention to prevent AGE formation and other types of damage caused by dicarboxyradicals, and to provide a protective effect to skin cells such as keratinocytes and fibroblasts from reactive carbonyl species.
The present invention provides a method for reducing protein, lipid, and DNA damage and change to skin cells by the administration of xcex1-amino-xcex2,xcex2-mercapto-xcex2,xcex2-dimethyl-ethane derivatives, e.g., D-penicillamine, which react with dicarbonyls to prevent direct damage to important cellular macromolecules. Methods of inhibiting DNA and skin cell photodamage are also disclosed.
The present invention also relates to a screening method for the identification of carbonyl scavengers via a rapid glycation system that proceeds independent of oxygen and therefore excludes identification of inhibitory compounds acting as antioxidants.